WO2023216799A1 - 一种人nkt细胞系及其应用 - Google Patents

一种人nkt细胞系及其应用 Download PDF

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WO2023216799A1
WO2023216799A1 PCT/CN2023/088019 CN2023088019W WO2023216799A1 WO 2023216799 A1 WO2023216799 A1 WO 2023216799A1 CN 2023088019 W CN2023088019 W CN 2023088019W WO 2023216799 A1 WO2023216799 A1 WO 2023216799A1
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cells
cell line
cell
nkt617
human
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朱学军
黎陈铖
刘细细
田芳
陈碧清
徐祖琼
姜鹏君
张玮光
杨箐
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江苏省中医院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A61K39/001112—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells

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  • the present invention relates to human cell lines, in particular to a human NKT cell line.
  • Natural Killer T cells are a special subset of T cells that express ⁇ TCR receptors ( ⁇ T cell receptors) to recognize antigens and exhibit typical NK cell characteristics, such as CD16, CD56, and Granzymes.
  • NKT cells can directly recognize lipid antigens presented by MHC class I molecule CD1d on the surface of target cells, or be activated by cytokines such as IL-2 and INF- ⁇ to rapidly respond.
  • Activated NKT cells can kill certain tumors and target cells infected by pathogens by secreting perforin, granzymes or through the Fas/Fas L pathway; they can also induce initial T cells to Th1 or Th2 cells by secreting IL-4 or IFN- ⁇ .
  • Differentiate participate in humoral immunity or cellular immune response, and enhance the body's anti-infection and anti-tumor effects.
  • NKT cells are also considered to be the bridge between innate immunity and adaptive immunity.
  • Type I NKT cells recognize lipid-based antigens presented by the ⁇ 2M-associated MHC class I molecule CD1d and are divided into two major categories: type I and type II NKT cells.
  • Type I NKT cells recognize the prototypical NKT cell lipid antigen ⁇ -galactosylceramide ( ⁇ -GalCer) and express a CD1d-restricted semi-invariant ⁇ TCR containing an invariant ⁇ -chain (mouse V ⁇ -J ⁇ 18, human V ⁇ 24- J ⁇ 18) is coupled to a limited ⁇ chain sequence (mouse V ⁇ 8, V ⁇ 7 and V ⁇ 2, human V ⁇ 11).
  • Type I NKT cells are relatively abundant in mice, accounting for 1% to 3% of T cells in most tissues and up to 50% of liver T cells. The proportion of human type I NKT cells is low, accounting for less than 1% of T cells in blood and liver. Humans have a greater number of type II NKT cells, which express different TCRs and have a wider range of lipid antigen specificities.
  • Extranodal Natural Killer/T-cell lymphoma is a highly aggressive subtype of non-Hodgkin lymphoma, usually CD56 + , intracellular CD3 + , intracellular toxicity markers Including granzyme B and TIA1 positivity.
  • NKT cells are genetically engineered to target them.
  • GD2 a molecule expressed on the surface of nearly all neuroblastoma cells, combines with genetically engineered chimeric antigen receptors and the secretion of IL-15 to maintain the activity of therapeutic cells in the immunosuppressive tumor microenvironment.
  • Preclinical studies have shown that this engineering design can increase the persistence of CAR-NKT cells and improve their effectiveness in the immunosuppressive tumor microenvironment, bringing hope for effective control of most solid tumors.
  • NKT Cells hold great promise as a new type of anti-cancer immunotherapy.
  • Chinese patent 201711386898 discloses a human NK/T cell line derived from a patient with chronic active EBV-infected lymphoproliferative disorder
  • Chinese patent 201210394807.9 discloses a human NK/T cell line derived from a patient with nasal angiocentric lymphoma.
  • NKT cell lines or T cell lines established from peripheral blood mononuclear cells (Hiroshi Nagata, 1 Ts ⁇ tom ⁇ N ⁇ mata, 1Akiyoshi Konno, Patho Logy International L 2001; 51:778–785). Most of them are transfected with EBV to obtain immortalized cell lines or are themselves infected with EBV. Therefore, providing an EBV-negative immortalized cell line is of great significance and unique value for various diseases and screening diagnostic molecules and drugs.
  • EBV-negative immortalized cell line is of great significance and unique value for NKTCL and screening of diagnostic molecules and drugs.
  • the present invention provides a human NKT cell line, named human NKT cell line 617, which is derived from the peripheral blood of NKTCL patients.
  • the patient is negative for EBV infection.
  • the established cell line EBV identification is also Tested negative.
  • the NKT cell line 617 of the present invention is derived from the peripheral blood of a female NKTCL patient.
  • the patient developed lymphadenopathy in the left neck region without obvious inducement, multiple lymphadenopathy and a large amount of ascites throughout the body, hepatosplenomegaly, and flow cytometry.
  • the test showed CD4 + , CD2 + , CD5 + , CD38 + , HLA-DR + , CD56 + , cCD3 + , mCD3 - , and TCR rearrangement was positive.
  • NKTCL is a lymphoma that is highly associated with EBV positivity, but this patient was negative for EBV infection, and the established cell line was also negative for EBV identification.
  • the NKT cell line 617 of the present invention is deposited at the China Type Culture Collection Center, the deposit number is: CCTCC NO: C2022121, the deposit date is May 9, 2022, and the deposit address is: Wuhan University, Wuhan, China.
  • This EBV-negative cell line can be continuously cultured in vitro for a long time. It has been cultured in vitro for more than two years and has been successfully monoclonal. It relies on low-dose IL-2 and can be massively expanded. It is a human NKT cell line for human NKT-related research and clinical adoptive immunity. Treatment provides new tools for experimental models.
  • NKT cells are not restricted by the highly polymorphic human leukocyte antigen (HLA), but by the monomorphic CD1d, so in contrast to traditional autologous CAR-T cell technology, CAR-NKT cells can achieve allogeneic , reducing the risk of graft-versus-host disease. Eliminate the need for personalized and patient-specific products that plague current CAR-T cell therapies.
  • Most of the NKT cells currently used for adoptive cellular immunotherapy are primary cells from healthy donors. Compared with primary NKT cells, genetic modification of mature and well-characterized homogeneous cell lines has many advantages: it can produce an "unlimited" number of NKT cells for CAR therapy; the consistency of the cell lines leads to higher CAR transduction efficiency, etc.
  • NKT617 The NKT cell line 617 (hereinafter referred to as NKT617) provided by the present invention eliminates the possibility of potential infection with EBV when used clinically as an allogeneic cell therapy. NKT cell lines have great prospects as a new type of anti-tumor cell immunotherapy.
  • Figure 1 shows the morphology of NKT617 cells at 100 ⁇
  • Figure 2 shows the results of Giemsa staining of NKT617 cells at 1000 ⁇ ;
  • Figure 3 shows the transmission electron microscope results of NKT617 cells (acceleration voltage 80kV, 2500 times magnification);
  • Figure 4 shows the scanning electron microscope results of NKT617 cells (acceleration voltage 3kV, 13.5mm length, 5000 times magnification);
  • Figure 5 shows the proliferation-dependent effects of NKT617 cells on IL-2 concentration
  • Figure 6 shows the results of partial spontaneous apoptosis of NKT617 over time in the absence of IL-2
  • Figure 7 shows the growth curve of NKT617 cells
  • Figure 8 shows the NKT617 karyotype
  • Figure 9 is a clonal rearrangement diagram of the NKT617TCR gene
  • FIG. 10 shows the results of NKT617 immunophenotyping analysis
  • Figure 11 shows the results of PCR detection of Mycoplasma in NKT617 cells
  • Figure 12 shows the results of Hoechst33342 staining of NKT617 cells
  • Figure 13 is a graph showing the detection results of the positive rate of anti-CD-CAR-NKT cells prepared by transfecting NKT617 cells;
  • Figure 14 shows the results of flow cytometry detection of CD-CAR-NKT killing Ramos cells in vitro.
  • Example 1 Establishment and characterization of NKT617 cell line.
  • NKTCL is a lymphoma that is highly associated with EBV positivity, but this patient was negative for EBV infection, and the established cell line was also negative for EBV identification.
  • lymphocytes After the cells were cultured for 72 hours, clear and vigorously growing lymphocytes were observed under the microscope. 500 ⁇ L of supernatant was aspirated every 2-3 days, and 1640 culture medium containing recombinant human IL-2 was added.
  • 1640 culture medium containing recombinant human IL-2 was added.
  • the cells in the wells of the cell culture plate are growing very vigorously, and there are cells growing in clusters. There are many translucent, round, lymphocytes of different sizes growing densely around it, and some of them are aggregated into bunches of grapes. grow. After two weeks of culture, carefully select a few wells where the cells are growing particularly vigorously. Gently pipet the cells in the wells and subculture them into T25 cell bottles.
  • the cells will grow well and at a fast growth rate and can be passaged in 2-3 days. once. After an interval of 2-3 days, observe the cell morphology under a microscope: take the cell culture plate and place it under an inverted microscope with an eyepiece of 10 ⁇ and an objective lens of 10 ⁇ .
  • Figure 1 shows the morphology of NKT617 cells (100 ⁇ ). The cells are translucent and round and grow vigorously.
  • NKT617 NKT cells of the present invention Line 617 is deposited at the China Type Culture Collection Center. The deposit number is: CCTCC NO: C2022121. The deposit date is May 9, 2022. The deposit address is: Wuhan University, Wuhan, China.
  • the culture medium used for NKT617 cell culture is: 1640 culture medium (Gibco product), containing 10% fetal bovine serum, recombinant human IL-2 (Roche) 50-100U/mL, and adding an appropriate amount of double antibodies (LIFE product).
  • Figure 1 is a picture of the optical morphology of cells at the 50th generation (100 ⁇ ). It can be seen that the cells are growing vigorously, with a clear background and few impurities. They are mainly translucent medium-sized lymphocytes, some growing as single cells, and some cells agglomerating with each other. Cells of varying sizes grow in clusters and can be passaged in 2-3 days.
  • Collect cells in the logarithmic growth phase add 2.5% glutaraldehyde, and fix overnight at 4°C. Rinse the sample three times with 0.1M, pH7.0 phosphate buffer for 15 minutes each time; then fix sample 1 with 1% osmic acid solution. -2h; pour away the fixative and rinse the sample three times with 0.1M, pH7.0 phosphate buffer, 15min each time; use gradient concentrations (including five concentrations of 50%, 70%, 80%, 90% and 95%) ethanol solution to dehydrate the sample, each Concentration treatment for 15 minutes, and then treatment with 100% ethanol for 20 minutes each time; finally transition to pure acetone treatment for 20 minutes; pure embedding agent is used to treat the sample overnight;
  • the samples were sliced in a Reichert ultramicrotome to obtain 70-90nm sections.
  • the sections were stained with lead citrate solution and uranyl acetate 50% ethanol saturated solution for 15 minutes each, and then were transmitted through the JEM-1230 model of Japan JEOL Company. Observed under electron microscope.
  • FIG. 3 The results of cell morphology observation under a transmission electron microscope are shown in Figure 3: The cells are oval in shape, with larger mitochondria clearly visible. Some of the nuclear chromatin is evenly distributed, and some are densely aggregated and unevenly distributed.
  • NKT617 cells were incubated with different concentrations of IL-2 from 0U/mL to 1000U/mL. Add 3,000 cells to each well of a 96-well plate, add 10ul CCK-8 (Beyotime) and incubate for 2 hours, and measure the absorbance at 450nm with a microplate reader.
  • CCK-8 Beyotime
  • NKT 617 cells were seeded in 6-well plates (2 ⁇ 10 5 cells/well) in RPMI-1640 containing 10% FBS. Add IL-2 (50U/mL) or not add IL-2, and culture the cells for several days. Cells were collected, centrifuged at 1000 ⁇ g for 5 minutes, and then resuspended in 500 ⁇ L Binding Buffer, followed by adding 5 ⁇ L AnnexinV-FITC and incubated in the dark at room temperature for 15 minutes. Add 5 ⁇ L PI before running on the machine. Use flow cytometer (BECKMAN COMELTER, NAVIOMS FLOW CYTOMETER) to detect FITC and PI fluorescence.
  • the total number of cells is 1.1 ⁇ 10 7 .
  • Add culture medium to 22 mL to make a cell suspension of 5.0 ⁇ 10 5 /mL. take 1 mL of cell suspension into a 30 mL culture bottle, add 4 mL of fresh culture medium, distribute it into 22 culture bottles, and place it in a 37°C, 5% CO2 incubator for culture. After 24 hours of culture, take 2 bottles of cells every day and count them using the trypan blue viable cell counting method.
  • the cell growth curve is shown in Figure 7.
  • the cell population doubling time was determined to be approximately 60 h.
  • the cells analyzed had complex karyotypes with multiple numerical and structural abnormalities.
  • NKT617 The karyotype of NKT617 is shown in Figure 8. Most of the tumor cells established have complex karyotypes, and the disease is prone to relapse and drug resistance is difficult to treat. This cell line provides a new tool and experimental model for human NKT-related research and clinical adoptive immunotherapy, and is of great significance and uniqueness for screening diagnostic molecules and drugs. value.
  • NKT617 cells showed clonal rearrangement of TCR ⁇ . It shows that NKT617 cells express T cell receptor (TCR).
  • TCR is a protein on the surface of T cells that is responsible for specifically recognizing antigen peptides that bind to MHC (major histocompatibility complex).
  • PBSA solution PBS plus 2% FBS
  • 400 ⁇ L of PBSA solution was added, and the cells were loaded and detected by flow cytometer (BECKMAN COMLTER, NAVIOMS FLOW CYTOMETER).
  • Figure 10 shows the results of NKT617 phenotype analysis: CD4 + CD8 + HLA-DR + CD45 + CD56 + CD16 - CD3 - cCD3 + CD25 + CD2 + CD38 + CD19 - CD5 - CD7 - CD80 - CD209 - CD83 - CD208 - CD20 - CD22 - C D138 - CD116 - CD163 - CD68 - CD86 - CD34 - CD10 - .
  • the high expression of CD45 indicates that NKT617 cells are of hematopoietic origin. They are negative for most of the B cell and myeloid markers tested, show the typical NK cell characteristic CD56, and also express T cell surface markers CD2, CD4, and CD8.
  • NKT617 is a T cell lineage with NK cell phenotype, and NKT617 was determined to be an NKT cell line.
  • Extract the DNA of NKT617 cell line configure the reaction system, NKT617 sample + negative control + positive control, and perform PCR amplification. Take 5 ⁇ L of PCR amplification product and use 1.5% agarose gel for electrophoresis detection.
  • RNA extraction kit (Shanghai Feijie, China) was used to extract RNA from NKT617 cells, and Taqman fluorescent probe was used for real-time fluorescence quantitative PCR. EBV primer sequence (5’-3’)
  • EBVforward cacaatgtcgtcttacaccattga
  • Epstein-Barr virus nucleic acid quantitative detection kit (Beijing Xinomedi Gene Detection Technology Co., Ltd.) was selected as positive quality control and negative quality control. Experimental results: No clear amplification curve was found for the NKT617 cell line (picture not shown).
  • the CD19scFv sequence FMC63 was synthesized with reference to the literature (China Cancer Biotherapy Impurities, 2018, 25(4), P389-393), and the bypass PCR technology was used to combine CD19-ScFv, hinge region CH2-CH3, CD137 transmembrane region and signal region, and intracellular
  • the CD3 ⁇ fragments that transmit signals are connected in series to form the second-generation CAR molecular structure of CD19-CAR.
  • the pCDH-CD19-CAR plasmid was obtained for subsequent lentivirus packaging.
  • CD19-CAR lentivirus was used to infect NKT617 cells, and the transfection efficiency was determined by detecting the level of GFP expression in NKT cells using fluorescence microscopy and flow cytometry.
  • Ramos cells are human lymphoma cells that express CD19 antigen. Therefore, Ramos cells are used as target cells and CD19-CAR-NKT is used as effector cells. The two cells are co-cultured at an effect-to-target ratio of 5:1. After killing for 4 hours in vitro, 7-AAD staining was used to determine the percentage of dead cells and determine the killing effect. The experimental results are shown in Figure 13. 72 hours after transfection, approximately 36.5% of NKT617 cells expressed GFP. The experimental results are shown in Figure 14. This shows that CD19-CAR-NKT cells prepared from NKT617 cells can effectively kill CD19-positive tumor cells.

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Abstract

一种人NKT细胞系及其在制备抗肿瘤细胞免疫治疗的制品上的应用，命名为NKT细胞系617，其保藏号为：CCTCC NO：C2022121，保藏日期为2022年5月9日。本发明的NKT细胞系617来源于一位女性NKTCL患者的外周血，此患者EBV感染呈阴性，建立的细胞系EBV鉴定亦呈阴性。该细胞系EBV阴性能长期在体外连续传代培养，目前已在体外培养两年余，单克隆成功，低剂量IL-2依赖可以大量扩增的人NKT细胞系，为人NKT相关研究及临床过继免疫治疗提供新的工具实验模型。

Description

一种人NKT细胞系及其应用 技术领域
本发明涉及人的细胞系，具体地涉及一种人NKT细胞系。
背景技术
自然杀伤T淋巴细胞(Natural Killer T cells cells，NKT细胞)是T细胞的一个特殊亚群，表达αβTCR受体(αβT细胞受体)以识别抗原并表现典型的NK细胞特征，如CD16、CD56以及颗粒酶。NKT细胞可直接识别靶细胞表面由MHCⅠ类分子CD1d提呈的脂质类抗原，或被IL-2、INF-γ等细胞因子激活，迅速产生应答。活化的NKT细胞可通过分泌穿孔素、颗粒酶或通过Fas/Fas L途径杀伤某种肿瘤和病原体感染的靶细胞；也可通过分泌IL-4或IFN-γ诱导初始T细胞向Th1或Th2细胞分化，参与体液免疫或细胞免疫应答，增强机体抗感染和抗肿瘤作用。NKT细胞也被认为是先天免疫和适应性免疫之间的桥梁。
NKT细胞识别由β2M相关的MHCⅠ类分子CD1d呈递的基于脂质的抗原，并分为两大类：Ⅰ型和Ⅱ型NKT细胞。Ⅰ型NKT细胞识别原型NKT细胞脂质抗原α-半乳糖神经酰胺(α-GalCer)并表达CD1d限制性半不变的αβTCR，包含不变的α-链(小鼠Vα-Jα18，人Vα24-Jα18)耦合到有限的β链序列(小鼠Vβ8，Vβ7和Vβ2，人Vβ11)。Ⅰ型NKT细胞在小鼠中相对丰富，占大多数组织中T细胞的1％～3％，以及在肝脏T细胞中高达50％。人类Ⅰ型NKT细胞占比较低，在血液和肝脏T细胞中不到1％。人类拥有更多数量的Ⅱ型NKT细胞，他们表达不同的TCR，具有更广泛的脂质抗原特异性。
结外自然杀伤T细胞淋巴瘤(Extranodal Natural Killer/T-cell lymphoma，NKTCL)是非霍奇金淋巴瘤的一种高度侵袭性的亚型，通常CD56+、胞内CD3+，胞内毒性标志物包括granzyme B和TIA1阳性。
2018年Cell Medica公司宣布全球首例儿童神经母细胞瘤患者已经成功接受该公司的在研的CAR-NKT疗法，基于该技术平台，通过对患者自身的NKT细胞进行基因工程改造，使其靶向GD2(一种几乎在所有神经母细胞瘤细胞表面均有表达的分子)，结合基因工程嵌合抗原受体和IL-15的分泌，在免疫抑制肿瘤微环境下维持治疗细胞的活性。临床前研究中已经显示，这种工程化设计可以增加CAR-NKT细胞的持久性并改善它们在免疫抑制性肿瘤微环境中的有效性，为实现对多数实体瘤的有效控制带来希望。NKT 细胞作为一种新型的抗癌细胞免疫治疗具有巨大的前景。
国内外少见NKT细胞系建立的报道，且大多转染EBV以获得永生化细胞株或本身已感染EBV。中国专利201711386898公开了一株源自人慢性活动性EBV感染淋巴细胞增殖症病人的人NK/T细胞系；中国专利201210394807.9公开了一株源自鼻部血管中心性NK/T细胞淋巴瘤患者的人NK/T细胞系。该细胞系表现为CD16-CD56-CD45-，非NK样；国外ADarji等人建立了一株来自髓系NK细胞急性白血病的NK/T细胞系。还有来自鼻腔NK/T细胞淋巴瘤患者(Nagata H,Konno A,Kimμra N,Zhang Y,Kimμra M,Demachi A,Sekine T,Yamamoto K,ShimizμN.BLood 2001,97:708–713)及CAEBV患者的外周血单个核细胞所建立的NKT细胞系或T细胞系(HiroshiNagata,1 TsμtomμNμmata,1Akiyoshi Konno,PathoLogyInternationaL 2001；51:778–785)。它们大多转染EBV以获得永生化细胞株或本身已感染EBV，因此提供一株EBV阴性的永生化细胞株对于各类疾病和筛选诊断分子以及药物具有重要意义和独特价值。
鉴于此，提供一株EBV阴性的永生化细胞株对于NKTCL和筛选诊断分子以及药物具有重要意义和独特价值。
发明内容
发明目的：本针对现有技术的不足，本发明提供一种人NKT细胞系，命名为人NKT细胞系617，其来源于NKTCL患者的外周血，患者EBV感染呈阴性，建立的细胞系EBV鉴定亦呈阴性。
本发明的NKT细胞系617来源一位女性NKTCL患者的外周血，患者无明显诱因出现左侧颈部区域淋巴结肿大，后全身多发淋巴结肿大及大量腹水，肝脾肿大，流式细胞学检测示CD4+、CD2+、CD5+、CD38+、HLA-DR+、CD56+、cCD3+、mCD3-，TCR重排阳性，结合临床诊断为NKTCL。NKTCL是一种与EB病毒阳性高度相关的淋巴瘤，但此患者EBV感染呈阴性，建立的细胞系EBV鉴定亦呈阴性。
本发明的NKT细胞系617保藏于保藏于中国典型培养物保藏中心，保藏号为：CCTCC NO：C2022121，保藏日为2022年5月9日，保藏地址为：中国武汉武汉大学。
该细胞系EBV阴性能长期在体外连续传代培养，目前已在体外培养两年余，单克隆成功，低剂量IL-2依赖可以大量扩增的人NKT细胞系，为人NKT相关研究及临床过继免疫治疗提供新的工具实验模型。
此外，NKT细胞不受高度多态性人类白细胞抗原(HLA)的限制，而是受单态性CD1d的限制，因此与传统的自体CAR-T细胞技术相反，CAR-NKT细胞可以实现同种异体，降低了移植物抗宿主病的风险。消除对困扰当前CAR-T细胞疗法的个性化和患者特异性产品需求。目前用于过继性细胞免疫疗法的NKT细胞多为健康供体的原代细胞。相比于原代NKT细胞，成熟且特征明确的同质细胞系基因改造有许多优势：产生“无限”数量的NKT细胞用于CAR治疗；细胞系的一致性导致CAR转导效率更高等。本发明提供的NKT细胞系617(下述简称为NKT617)消除了作为同种异体细胞疗法临床使用时，潜在感染EBV的可能。NKT细胞系作为一种新型的抗肿瘤细胞免疫治疗具有巨大的前景。
附图说明
图1为100×下NKT617细胞形态；
图2为1000×下NKT617细胞吉姆萨染色图片结果图；
图3为NKT617细胞透射电镜结果(加速电压80kV，2500倍放大)；
图4为NKT617细胞扫描电镜结果(加速电压3kV，13.5mm长度5000倍放大)；
图5为NKT617细胞对IL-2浓度的增殖依赖性的影响；
图6为无IL-2条件下，NKT617随时间发生部分自发凋亡的结果图；
图7为NKT617细胞的增长曲线图；
图8为NKT617染色体核型图；
图9为NKT617TCR基因的克隆性重排图；
图10为NKT617免疫表型分析结果；
图11为NKT617细胞支原体PCR检测结果图；
图12为NKT617细胞Hoechst33342染色结果图；
图13为转染NKT617细胞制备的抗CD-CAR-NKT细胞阳性率检测结果图；
图14为流式细胞术检测CD-CAR-NKT体外杀伤Ramos细胞结果图。
具体实施方式
下面结合附图和具体实施方式对本发明做更进一步的具体说明，本发明的上述和/或其他方面的优点将会变得更加清楚。
实施例1 NKT617细胞系的建立和表征。
(1)细胞系的建立
1.1标本来源：
取自一名患有NKTCL女性患者的外周血，肝素抗凝。经患者及家属同意，签订知情同意书。该患者无明显诱因出现左侧颈部区域淋巴结肿大，后全身多发淋巴结肿大及大量腹水，肝脾肿大，流式细胞学检测示CD4+、CD2+、CD5+、CD38+、HLA-DR+、CD56+、cCD3+、CD3-，TCR重排阳性，结合临床诊断为NKTCL。NKTCL是一种与EB病毒阳性高度相关的淋巴瘤，但此患者EBV感染呈阴性，建立的细胞系EBV鉴定亦呈阴性。
1.2外周血单个核细胞分离：
无菌抽取患者外周血2mL，用淋巴细胞分离液进行密度梯度离心，2000rpm，室温20℃，离心20min。吸取中界层面的单个核细胞，经PBS液洗2遍，1500rpm，室温20℃，离心10min，加入新鲜完全的1640培养液，后将2mL细胞悬液分装至24孔培养板内，并添加50-100U的重组人IL-2，在37℃、5％CO2培养箱中进行培养。
1.3细胞的培养建系
细胞培养72小时后，镜下观察可见透亮而生长旺盛的淋巴细胞，每间隔2-3天吸掉500μL上清，加入含有重组人IL-2的1640培养液。一周以后，在镜下观察，可见细胞培养板孔内细胞生长特别旺盛，有细胞成团生长现象，周边有许多透亮、圆形、大小不等的淋巴细胞密集生长，有的凝聚成葡萄串样生长。培养二周后，仔细挑选出几个细胞生长特别旺盛的孔，将孔内细胞轻轻吹打后传代至T25细胞瓶中培养，随后细胞生长状况好，且生长速率快，2-3天可以传代一次。间隔2-3天后显微镜观察细胞形态：取细胞培养板置于倒置显微镜下，目镜10×，物镜10×。图1所示为NKT617细胞形态(100×)，细胞呈透亮圆形，生长旺盛。
该细胞已连续稳定生长2年以上，经液氮冻存后复苏，细胞状态好，存活率达95％以上，将该细胞命名为NKT细胞系617(下文简称为NKT617)。本发明的NKT细胞 系617保藏于保藏于中国典型培养物保藏中心，保藏号为：CCTCC NO：C2022121，保藏日为2022年5月9日，保藏地址为：中国武汉武汉大学。
NKT617细胞培养所用的培养液为：1640培养液(Gibco产品)，内含10％胎牛血清、重组人IL-2(Roche)50-100U/mL，并加适量双抗(LIFE产品)。
(2)细胞学形态的观察
2.1光镜下的细胞形态学观察
2.1.1培养的活细胞形态
取传代培养的细胞，在光学显微镜下(日本OLympus IMT-2倒置显微镜)观察活细胞生长情况。图1为第50代细胞光学形态图片(100×)，可见细胞生长旺盛，背景清晰，杂质少见，以透亮的中等大小的淋巴细胞为主，有的呈单细胞生长，有的细胞相互凝集成大小不等的细胞团样生长，2-3天可以传代。
2.1.2吉姆萨染色法细胞涂片
取对数生长期的细胞100μL，置于细胞离心涂片器上，在Thermo SCIENTIFIC涂片机上以1000rpm，室温离心1min，悬浮细胞离心沉积涂抹在玻片上，形成单层细胞涂片。用苏木精和伊红染色(Richard Allen Scientific)，取玻片置于倒置显微镜下，目镜10×，物镜100×。图2所示为NKT617细胞形态(1000×)，细胞呈类圆形，细胞核染成深紫色。
2.2电镜下的细胞形态学观察
2.2.1透射电镜下细胞形态观察
收集对数生长期的细胞加入2.5％的戊二醛，4℃固定过夜，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；再用1％的锇酸溶液固定样品1-2h；倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；用梯度浓度(包括50％，70％，80％，90％和95％五种浓度)的乙醇溶液对样品进行脱水处理，每种 浓度处理15min，再用100％的乙醇处理一次，每次20min；最后过度到纯丙酮处理20min；纯包埋剂处理样品过夜；
将经过渗透处理的样品包埋起来，70℃加热过夜，即得到包埋好的样品。样品在Reichert超薄切片机中切片，获得70-90nm的切片，该切片经柠檬酸铅溶液和醋酸双氧铀50％乙醇饱和溶液各染色15min，即可在日本JEOL公司的JEM-1230型透射电镜下观察。
透射电镜下细胞形态观察结果，如图3所示：细胞呈卵圆形，清晰可见较大的线粒体，核染色质有的均匀分布，有的聚集浓密，分布不均。
2.2.2扫描电镜下细胞形态观察
收集对数生长期的细胞，Hank’s液洗二遍，最后一遍1500rpm，离心7min，弃去上清液，加入2.5％的戊二醛，4℃固定过夜，0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；用1％的锇酸溶液固定样品1-2h；用梯度浓度(包括50％，70％，80％，90％和95％五种浓度)的乙醇溶液对样品进行脱水处理，再用乙醇与醋酸异戊酯的混合液(V/V＝1/1)处理样品30min，再用纯醋酸异戊酯处理样品1-2h。
临界点干燥；镀膜，观察。将处理好的样品在荷兰PhiLips公司的XL30型环境扫描电镜中观察。如图4所示：细胞呈类圆形，细胞表面有大小不等的凹陷，有的细胞凝聚在一起生长。
(3)细胞对IL-2增殖依赖性
3.1细胞增殖：
NKT617细胞用不同浓度IL-2从0U/mL到1000U/mL孵育。96孔板每孔加入3000个细胞，加入10ul CCK-8(碧云天Beyotime)孵育2h，用酶标仪测定在450nm处的吸光度。
实验结果如图5所示：细胞呈IL-2依赖增殖。
3.2细胞凋亡
将NKT 617细胞接种在含有10％FBS的RPMI-1640的6孔板(2×105细胞/孔)中。加入IL-2(50U/mL)或者不加入IL-2，培养细胞数天。收集细胞，以1000×g离心5分钟，然后重悬于500μL Binding Buffer中，随后加入5μL AnnexinV-FITC，在室温下黑暗中孵育15分钟。上机前补加5μL PI。用流式细胞仪(BECKMAN COΜLTER，，NAVIOMS FLOW CYTOMETER)检测FITC、PI荧光。
实验结果如图6所示：在无IL-2条件下，48h后NKT617的细胞凋亡率(19.8％)高于含50U/mL IL-2培养的NKT617(1.9％)，72h后无IL-2培养的NKT617细胞凋亡率(22.7％)高于含50U/mL IL-2培养的NKT617(2.5％)。表明在无IL-2条件下，NKT617细胞发生部分自发凋亡，呈IL-2依赖生长。
(4)细胞生长曲线
取对数生长期的生长良好传代细胞，采用台盼兰活细胞计数法计数定量，细胞总数为1.1×107个，用培养液加至22mL，制成5.0×105/mL的细胞悬液，取1mL细胞悬液于30mL培养瓶中，再加4mL新鲜培养液，共分装在22个培养瓶中，置于37℃，5％CO2的培养箱中培养。培养24小时以后，每天取2瓶细胞，用台盼兰活细胞计数法计数。
细胞生长曲线如图7所示。测定细胞群体倍增时间约为60h。
(5)细胞遗传学分析
取对数生长期的传代细胞，在试验的前一天传代培养，用10mL 1640培养液(内含IL-2 50U/mL)悬浮培养在37℃5％CO2培养箱中培养24小时。使用秋水仙素、低渗液、甲醇/乙酸固定和Wright染色。在油镜下进行计数，共计数100个分裂象，进行分析。
结果：所分析10个细胞中：
7个为47,XX,add(9)(p24),+11,？der(11)t(11；19)(q23；p13),-13,13p+,add(15)(q26),
del(16)(q22),i(17)(q10),-21,22p+c,+M1？13q-,+M2,dmin.
1个为47,idem,1q-.
1个为48,idem,9p-,+？der(11)t(11；19).
1个为63,XX,+1,+1,del(1)(p34),+2,+2,+3,+5,+8,add(9)(p24),+10,+11,+11,？der 11),+？der(11),+12,-13,+14,del(16)(q22),+19,+20,+20,-21,-22,+M1×2？13q-,+M2.
所分析细胞均为伴有多种数目和结构异常的复杂核型。
NKT617染色体核型如图8所示。建系的肿瘤细胞大多为复杂核型，疾病易于复发，耐药难治，此细胞系为人NKT相关研究及临床过继免疫治疗提供新的工具实验模型，为筛选诊断分子及药物具有重要意义和独特价值。
(6)细胞TCR基因重排
用DNA抽提试剂盒(上海飞捷，中国)提取生长状态良好的NKT617细胞的DNA，与TCR特异性引物进行第一轮PCR扩增；960μL的Hi-Di溶液与40μL的GeneScanTM-500LIZ混合，短甩，离心后分装入96孔测序板中。每个反应孔中9μL上述溶液，对照用等量去离子水补齐，进行第二轮PCR扩增。使用3730仪器读取样本。
实验结果如图9所示，NKT617细胞呈TCRβ克隆性重排。表明NKT617细胞表达T细胞受体(T cell receptor，TCR)，TCR是T细胞表面，负责特异性识别与MHC(主要组织相容性复合体)结合的抗原肽的蛋白。
(7)细胞免疫表型分析
取约1×106对数生长期的传代细胞，收集于15mL的离心管中，用台盼兰活细胞计数，细胞经PBSA液(PBS加2％FBS)洗2遍后，加入200μL人血清孵育10min，2000rpm离心2min，收集于1.5mL的离心管用100μL PBS，分成5管，2000rpm离心2min去除上清，加入2μL相应的抗体，避光冰上放置30分钟。胞经PBSA液(PBS加2％FBS)洗2遍后，再加400μL PBSA液，经流式细胞仪(BECKMAN COMLTER,NAVIOMS FLOW CYTOMETER)上样检测。
图10所示NKT617表型分析结果:CD4+CD8+HLA-DR+CD45+CD56+CD16-CD3-cCD3+CD25+CD2+CD38+CD19-CD5-CD7-CD80-CD209-CD83-CD208-CD20-CD22-C D138-CD116-CD163-CD68-CD86-CD34-CD10-。CD45高表达表明NKT617细胞是造血来源，其对所检测的大多数B细胞和髓系标记物表达均为阴性，表现典型的NK细胞特征CD56，也表达T细胞表面标志物CD2、CD4、CD8。
结合上述NKT617细胞表现TCRβ克隆性重排，表明NKT617是具有NK细胞表型的T细胞谱系，确定NKT617为NKT细胞系。
(8)细胞支原体检测
8.1 PCR检测法
提取NKT617细胞系DNA，配置反应体系，NKT617样本+阴性对照+阳性对照，进行PCR扩增。取5μLPCR扩增产物，使用1.5％琼脂糖凝胶进行电泳检测。
结果如图11所示，NKT617样品的上清液中未检测到目标条带，表明NKT617样品支原体检测呈阴性。
8.2.荧光染料法(Hoechst33258)检测
加入NKT617样品3倍体积的Hoechst33258染色液，混匀。室温放置3-5分钟。吸除染色液，用生理盐水洗涤2-3次，每次3-5分钟。直接在荧光显微镜下观察或封片后荧光显微镜下观察。
实验结果如图12所示：阳性对照荧光显微镜下除细胞外，可见大小不等、不规则的荧光着色颗粒(如图B，改编自Hoechst33258染色说明书，碧云天)。NKT617细胞内未见明显荧光着色颗粒(如图A 200×)。表明NKT617细胞未感染支原体。
(9)细胞系EBV病毒检测
利用RNA抽提试剂盒(上海飞捷，中国)提取NKT617细胞RNA，使用Taqman荧光探针进行实时荧光定量PCR。EBV引物序列(5’-3’)
EBVforward：cacaatgtcgtcttacaccattga，
EBVreverse：aggtccttaatcgcatccttca。
选用EB病毒核酸定量检测试剂盒(北京鑫诺美迪基因检测技术有限公司)作为阳性质控和阴性质控。实验结果：NKT617细胞系未见明确扩增曲线(未显示图)。
(10)CD19-CAR-NKT的体外构建及对靶细胞的杀伤能力验证
参照文献(中国肿瘤生物治疗杂质，2018,25(4)，P389-393)合成CD19scFv序列FMC63，利用搭桥PCR技术将CD19-ScFv、铰链区CH2-CH3、CD137穿膜区和信号区以及胞内传递信号的CD3ζ片段依次串联，形成第二代CAR分子结构的CD19-CAR。获得pCDH-CD19-CAR质粒，用于后续慢病毒包装。利用CD19-CAR慢病毒感染NKT617细胞，通过荧光显微镜及流式细胞术检测NKT细胞表达GFP水平确定转染效率。Ramos细胞是表达CD19抗原的人淋巴瘤细胞，因此以Ramos细胞为靶细胞，CD19-CAR-NKT为效应细胞，按效靶比5:1将这两种细胞共培养，体外杀伤4h后，通过7-AAD染色确定死细胞百分比，确定杀伤效果。实验结果如图13所示。转染72h后，约有36.5％的NKT617细胞表达GFP。实验结果如图14所示。说明由NKT617细胞制备的CD19-CAR-NKT细胞能够有效杀伤CD19阳性肿瘤细胞。

Claims (4)

  1. 一种人NKT细胞系，命名为NKT细胞系617，其保藏于中国典型培养物保藏中心，保藏号为：CCTCC NO：C2022121，保藏日为2022年5月9日。
  2. 根据权利要求1所述的人NKT细胞系，其特征在于，所述人NKT617细胞系来源于NKTCL患者的外周血，患者EBV感染呈阴性。
  3. 根据权利要求1所述的人NKT细胞系，其特征在于，建立的细胞系EBV鉴定呈阴性。
  4. 权利要求1所述的人NKT细胞系在制备抗肿瘤细胞免疫治疗的制品上的应用。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016513A1 (en) * 2001-08-13 2003-02-27 Junjiro Tsuchiyama Epstein-barr virus-negative nk cell line
CN102876632A (zh) * 2012-10-17 2013-01-16 浙江省肿瘤医院 人nk/t细胞系
WO2018056736A1 (ko) * 2016-09-23 2018-03-29 주식회사 에스엘바이젠 신규 자연살해 세포주 및 그의 용도
CN108220237A (zh) * 2017-12-20 2018-06-29 中国科学院遗传与发育生物学研究所 一种人的nk/t细胞系
CN115094034A (zh) * 2022-05-11 2022-09-23 江苏省中医院 一种人nkt细胞系及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220236B (zh) * 2017-12-20 2020-06-30 中国科学院遗传与发育生物学研究所 一种人的nk细胞系

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016513A1 (en) * 2001-08-13 2003-02-27 Junjiro Tsuchiyama Epstein-barr virus-negative nk cell line
CN102876632A (zh) * 2012-10-17 2013-01-16 浙江省肿瘤医院 人nk/t细胞系
WO2018056736A1 (ko) * 2016-09-23 2018-03-29 주식회사 에스엘바이젠 신규 자연살해 세포주 및 그의 용도
CN108220237A (zh) * 2017-12-20 2018-06-29 中国科学院遗传与发育生物学研究所 一种人的nk/t细胞系
CN115094034A (zh) * 2022-05-11 2022-09-23 江苏省中医院 一种人nkt细胞系及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ASIF SAMIA, BEGEMANN MADELINE, BENNETT JOSEPH, FATIMA RAWISH, MASOOD ASHIQ, RAZA SHAHZAD: "Pembrolizumab in newly diagnosed EBV‑negative extranodal natural killer/T‑cell lymphoma: A case report", MOLECULAR AND CLINICAL ONCOLOGY, SPANDIDOS PUBLICATIONS, GR, vol. 10, no. 3, 1 March 2019 (2019-03-01), GR , pages 397 - 400, XP093107298, ISSN: 2049-9450, DOI: 10.3892/mco.2019.1805 *

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